Thermophile gene transfer

ABSTRACT

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan tr2 ). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu -  Km r  transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

This application is a continuation of application Ser. No. 08/265,522,filed on 24 Jun., 1994, now abandoned.

FIELD OF THE INVENTION

The instant invention is related to the field of thermophilicmicroorganisms the stable gene transfection in and protein expressionthereof, and in the genetic thermostabilization of proteins.

BACKGROUND OF THE INVENTION

Extreme thermophilic microorganisms such as Thermus, thrive inhigh-temperature environments that are lethal to other known forms oflife. Fortunately, apart from their higher growth temperaturerequirement, they can be handled in the laboratory much like E. coli.Enzymes from thermophiles are thermostable and are therefore used inindustrial processes that benefit from a high reaction temperature.Also, these enzymes have become widely used in molecular geneticresearch, for example, in the development and application of thepolymerase chain reaction (PCR).

One area of particular interest in the field of thermophile research isthe determination of molecular mechanisms underlying enzymaticthermostability. Ultimately, a better understanding of this phenomenonwill allow mesophilic proteins to be rationally converted tothermostable proteins for industrial applications. Many groups haveattempted to engineer thermostability into proteins through in vitrorational design approaches (Perry and Wetzel, 1984, Science 226:555-557;Sauer, et al., 1986, Biochemistry 25:5992-8; Pantoliano, et al., 1987,Biochemistry 26:2077-82; Meng, et al., 1993, Bio/Technology11:1157-1161) or through homology comparison and domain fusion ofrelated proteins (Onodera, et al., 1991, J Biochem 109:1-2; Barany, etal., 1992, Gene 112:3-12; Politz, et al., 1993, Eur J Biochem216:829-34; Lee, et al., 1993, J Bacteriol 175:5890-8). Theseapproaches, however, often require either a three dimensional proteinstructure or a series of related proteins. For proteins which have notbeen well characterized, random mutagenesis can be a powerful tool ifthe proper selection or screen can be applied (Matsumura and Aiba, 1985,J Biol Chem 260:15298-15303; Liao, et al., 1986, Proc Natl Acad Sci USA83:576-580; Kajiyama and Nakano, 1993, Biochemistry 32:13795-9; Arnold,1993, Faseb J 7:744-9). The instant invention provides a genetic processfor the insertion of exogenous protein coding sequences into, and directselection of thermostable variants of mesophilic enzymes. Other"thermo-genetic" processes, were attempted by Liao (Liao et al., 1986,Proc. Natl. Acad. Sci. USA 83:576-580), EP Patent application 0 138 075,and by Matsumura (Matsumura et al., 1985, J Biol. Chem.260:15298-15303).

The concept of "thermo-genetics" consists of a method for introducing agene of interest into a thermophile followed by a temperature-shift toselect for temperature-resistant mutations in the corresponding proteinof interest. The model thermo-genetic systems (EP Patent application 0138 075; Matsumura, 1985; Liao, 1986) used the mesophilickanamycin-resistance gene (kan) on a multicopy plasmid in the moderatethermophile Bacillus stearothermophilus. The kan gene was firstintroduced into B. stearothermophilus at the lowest permissibletemperature of growth, 47° C. Two consecutive thermal shifts, first to63° C. and then to 69° C., resulted in two correspondingthermo-stabilizing mutations, producing the double mutant allele,designated here as kan^(tr2). At this point the upper limit forpermissible growth had been reached, creating a barrier to furtherselections for temperature-resistant mutations. Matsumura also performeda related series of experiments generating the same two mutants inparallel (Matsumura et al., 1986, Nature 323:356-358) and later showedthey could be combined with an additive result.

While other host-vector systems have been developed for Thermusthermophilus, a closely related thermophile, they all have deficienciesin their ability to be used in a thermostabilization process and instable integration of exogenous genes into Thermus. Two plasmid-basedsystems use multicopy plasmids with an unstable copy number which caninterfere with mutant selection (Mather and Fee, 1992, Appl EnvironMicrobiol 58:421-425; Lasa, et al., 1992, J Bacteriol 174:6424-6431).The multicopy nature of these systems do not ideally lend themselves tothermostabilization of genes since many copies of the gene of interestare present, and can mask any desired mutations which may occur. Inaddition, reports with plasmid-based systems in Thermus indicate thatthe plasmids are very unstable, that copy number varies widely, and thatgene duplication and amplification can occur, making them very difficultto use. Another approach which used an insertional mutagenesis systemwas developed by Lasa et. al. (Lasa, et al, 1992, Molec Microbiol6:1555-1564) but unfortunately caused a debilitating phenotype in thehost organism.

In Lasa's insertional mutagenesis system, the kan^(tr2) was inserted insingle copy into a highly-expressed (slpA) region of the chromosome foruse in chromosomal insertion strategy (Lasa et al. 1992a, J. Molec.Microbiol. 6, 1555-1564; Lasa et al. 1992b, J. Bacteriol. 174,6424-6431). This system used the slpA gene which codes for an abundantcell surface protein and therefore was likely to be highly expressed. Ahigh expression site was originally a logical choice for testing thefeasibility of a single-copy system.

Unfortunately, insertion into slpA results in debilitating growth andmorphology phenotypes making it difficult to use the plasmid system.

References which define the background of the invention, but which arenot necessarily prior art to the instant invention are as follows. Thereferences cited herein, above and below are hereby incorporated byreference in their entirety.

Sen & Oriel (1990) Transfer of transposon Tn916 from Bacillus subtilisto Thermus aquaticus, FEMS Microbiology Letters 67:131-134, teach theuse of the Streptococcus transposon Tn916, carrying tetracyclineresistance for conjugal transfer into Thermus aquaticus via Bacillussubtilis. This was found to be effective at 48° C. and 55° C. The actualinsertion site is unknown.

Koyama et al. (1990) A plasmid vector for an extreme thermophile,Thermus thermophilus, FEMS Microbiology Letters 72:97-102, teach aThermus-E. coli shuttle vector carrying a tryptophan synthetase gene(trpB). This cryptic plasmid pTT8, was able to transform Thermusthermophilus. The authors point out that a plasmid vector carrying trpBAwas not suitable for selection since the cloned DNA fragment recombinedwith the chromosomal counterpart at high frequency.

Koyama & Furukawa (1990) Cloning and Sequence Analysis of TryptophanSynthetase Genes of an Extreme Thermophile, Thermus thermophilus HB27:Plasmid Transfer from Replica-Plated Escherichia coli RecombinantColonies to Competent T. thermophilus Cells, J. of Bacteriology172:3490-3495, disclose nucleotide sequences for trpBA genes, their usein plasmids and expression in E. coli under the control of the lacpromoter.

Koyama et al. (1986) Genetic Transformation of the Extreme ThermophileThermus thermophilus and of Other Thermus spp., J. of Bacteriology166:338-340, discuss the conditions for optimal transformation withexogenous DNA. The use of Thermus thermophilus HB27 did not requirechemical treatment to induce competence, although the addition of Ca⁺²and Mg⁺² was optimal. The optimal conditions were found to be 70° C.with a 60 minute incubation, pH 6 to 9.

Borges & Bergquist (1993) Genomic Restriction Map of the ExtremelyThermophilic Bacterium Thermus thermophilus HB8, J. of Bacteriology175:103-110, teach the use of Thermus thermophilus HB8, which carriestwo cryptic plasmids, pTT8 and pVV8 was examined. A genomic restrictionmap was generated, 16 genes located on the map.

Matsumura et al (1984) Enzymatic and Nucleotide Sequence Studies of aKanamycin-Inactivating Enzyme Encoded by a Plasmid from ThermophilicBacilli in Comparison with That encoded by Plasmid pUB110, J. ofBacteriology 160:413-420, teach the a Kanamycin resistance gene from athermophilic bacteria plasmid pTB913 was found to differ by only onebase pair in the middle of the gene, from that of a mesophilicStaphylococcus aureus plasmid pUB110. The change was a cytosine(pUB) toadenine(pTB) at base position +389, which led to a threonine to lysinechange at position 130.

Matsumura & Aiba (1985) Screening for Thermostable Mutant of KanamycinNucleotidyltransferase by the Use of a Transformation System for aThermophile, Bacillus stearothermophilus, J. of Biological Chemistry260:15289-15303, disclose a structural gene for kanamycinnucleotidyltransferase that was cloned into the single-strandedbacteriophage M13 and then subjected to hydroxylamine mutagenesis. Themutagenized gene was then recloned into a vector plasmid pTB922 and usedto transform Bacillus stearothermophilus to select for improved enzymethermostability. A temperature shift from 55° C. to 61° C. was used forselection. Two types of mutations were found, at position 80 anaspartate to tryptophan, and at position 130 a threonine to lysine.These were found stable up to 65° C. The kan gene came from pUB110.

Matsumura et al. (1986) Cumulative effect of intragenic amino-acidreplacements on the thermostability of a protein, Nature 323:356-358,teach improved thermostability of kanamycin nucleotidyltransferase(KNTase) was shown to be due to the Asp80 to Tyr(Y80) and Thr130 toLys(K130) mutation. This also correlated with increased resistance toproteolysis. Catalytic activity was also measured at varioustemperatures and it was found that the activities deteriorate slightlyas thermostability increases, but the optimal temperature is shiftedupwards. It is thought that increased hydrogen bonding and hydrophobicinteractions act as forces to stabilize the enzyme.

Liao et al. (1986) Isolation of a thermostable enzyme variant by cloningand selection in a thermophile, PNAS USA 83:576-580, teach a kan genetransferred via shuttle vector into B. stearothermophilus and selectedfor at 63° C. The shuttle plasmid was passed through the E. coli mutD5mutator strain and introduced by transformation. The vector combined thekan gene from pUB110 with a putative thermostable origin of replicationfrom pBST1, isolated from a kanamycin-sensitive strain NRRL1102.

Lasa et al. (1992a) Development of Thermus-Escherichia Shuttle Vectorsand Their Use for Expression of the Clostridium thermocellum celA Genein Thermus thermophilus, J. of Bacteriology 174:6424-6431, teach theself-selection of undescribed origins of replication from crypticplasmids from uncharacterized Thermus spp. and Thermus aquaticus areisolated and cloned into E. coli vectors. Plasmids were constructed withthese origins, pLU1 to pLU4 from T. aquaticus, and pMY1 to pMY3 fromThermus spp. The plasmids then had a modified form of the cellulase gene(celA) from Clostridium thermocellum and were expressed in E. coli withthe signal peptide from the S-layer gene from T. thermophilus.Transformation back into T. thermophilus allowed for expression at 70°C.

Lasa et al. (1992b) Insertional mutagenesis in the extreme thermophiliceubacteria Thermus thermophilus HB8, Molecular Microbiology 6:1555-1564,teach the transcription and translation signals from the slpA gene fromThermus thermophilus HB8 used to express a thermostable kan gene. After48 hours at 70° C., two isolates were obtained.

Faraldo et al. (1992) Sequence of the S-Layer Gene of Thermusthermophilus HB8 and Functionality of Its Promoter in Escherichia coli,J. of Bacteriology 174:7458-7462, disclose the S-layer gene slpA ,sequenced and the function in E. coli described.

Mather & Fee (1992) Development of Plasmid Cloning Vectors for Thermusthermophilus HB8: Expression of a Heterologous, Plasmid-Borne KanamycinNucleotidyltransferase Gene, Applied and Envior. Microbiology 58:421-425

A plasmid cloning vector is disclosed which uses the kan gene insertedrandomly into a cryptic multicopy plasmid (pTT8) isolated from T.thermophilus.

Nagahari et al. (1980) Cloning and expression of the leucine gene fromThermus thermophilus in Escherichia coli, Gene 10:137-145, describe theThermus thermophilus leu locus cloned into E. coli and expressed. Theplasmid pBR322-T.leu hybrid plasmid was constructed to encode the β-IPMdehydrogenase activity (leuB), the optimal temperature of which was 80°C. Experiments suggest that there is a promoter that may be used in E.coli.

Tanaka et al. (1981) Cloning of 3-Isopropylmalate Dehydrogenase Gene ofan Extreme Thermophile and Partial Purification of the Gene Product,Biochem. 89:677-682, demonstrate the cloning into E. coli, proteinproduction, and heat-treatment purification of Thermus thermophilus3-IPM.

Croft et al. (1987) Expression of leucine genes from an extremelythermophilic bacterium in Escherichia coli, Molec. Gen. Genet.210:490-497, describe the promoter for the leu BCD genes in Thermusthermophilus HB8. The structural similarity with known leu genes isexamined. Thermus DNA failed to complement E. coli leuA mutants. PerhapsleuA is not functional.

Yamada et al (1990) Purification, Catalytic Properties, and ThermalStability of Threo-Ds-3-Isopropylmalate Dehydrogenase Coded by leuB Geneform an Extreme Thermophile, Thermus thermophilus Strain HB8, J.Biochem. 108:449-456, demonstrate the product of leuB from Thermusthermophhilus as expressed in E. coli by plasmid. The enzyme waspurified using heat treatment.

Kirino & Oshima (1991) Molecular Cloning and Nucleotide Sequence of3-Isopropylmalate Dehydrogenase Gene (leuB) from an Extreme Thermophile,Thermus aquaticus YT-1, J. Biochem. 109:852-857, here the gene encodingT. aquaticus leuB was cloned into E. coli and expressed.

Imada et al. (1991) Three-dimensional Structure of a Highly ThermostableEnzyme, 3-Isopropylmalate Dehydrogenase of Thermus thermophilus at 2.2 ÅResolution, J. Mol. Bio. 222:725-738, desribe the 3D structure of IPMDHfrom Thermus thermophilus has been determined and refined to 2.2 Åresolution. The dimeric form of IPMDH is crucial to function.

Onodera et al. (1991) Crystallization and Preliminary X-Ray Studies of aBacillus subtilis and Thermus thermophilus HB8 Chimeric3-Isopropylmalate Dehydrogenase, J. Biochem. 109:1-2, teach a chimericgene fusing the Bacillus subtilis and Thermus thermophilus genesencoding for IPMDH, cloned into E. coli.

Liao et al., European Patent Application 0 138 075 A1 published24.04.85, discloses the use of plasmids for transforming thermophilicbacteria. A method for isolating thermostable promoters, a method forselecting thermostable variants of gene products of cloned genes inthermophilic hosts using the plasmids of the invention. Such plasmidsas:

pBST1, 80 kb cryptic single copy plasmid isolated from B.stearothermophilus at 70° C.

pBST2, 1.4 kb, O_(R) of pBST1 & kan^(R) of pUB110, grows at 70° C., kanup to 47° C. 3 copy.

pBST2-6, pBST2 with oligonucleotide linker to form HindIII site. kan<55° C.

pBST2-6TK, variant of pBST2-6, kan activity up to 66° C.

pSHW9, chloramphenicol^(R) CATfrom pC194, in pBR322, amp^(R), pBR322OR+pC194.

pBST8, pBST2-6+pSHW9, "Shuttle vector"

pCV1, pSHW9 with promoter substituted by polylinker site.

pCV3, pCV1 missing NarI site.

pBST110, pCV3+pBST2-6, shuttle vector, no CAT production.

pRMS10, shotgun clone into pBST110, a promoter from B.stearothermophilus.

Lacey, R. W. and I. Chopra. Genetic studies of a multiresistant strainof Staphylococcus aureus. J. Med. Microbiol. 7:285-297, 1974, discribesplasmid pUB110 which contains the Kan gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the physical map of pVUF10.5 composed of the KpnI fragmentfrom pVUF10 containing pyrE gene (grey) cloned into the KpnI site ofpUC19 (black). The location and direction of the bla gene conferringampicillin resistance and the pyrE gene from T. flavus are shown witharrows.

FIG. 2 is the nucleic acid sequence (SEQ ID NO:1) of the pyrE gene fromT. flavus. The DNA sequence which complements an E. coli pyrE mutationand the open reading frame thought to encode the pyrE gene of Thermusflavus are shown. The standard three-letter amino acid sequences areused.

FIGS. 3A-3B are plasmid maps. (A) pTG100, derived from the E. colivector pTZ18R digested with BamHI (open arrows and bars) and containinga 3 kb Sau3AI fragment of T. flavus chromosomal DNA encoding the leuBgene (shaded arrow) and flanking sequences (shaded bars). (B) Map ofpTG100kan^(tr2), a plasmid derived from pTG100, contains the kan^(tr2)gene (solid dark arrow) inserted into the blunted NcoI site of the leuBgene (shaded arrow). The plasmid pTG100kan, described in the text, isidentical to pTG100kan^(tr2) except it contains the wild-type allele ofthe kanamycin-resistance gene, kan, inserted into the blunted NcoI siteof pTG100.

FIG. 4 is a comparative analysis of the restriction maps of leuB genesfrom three different Thermus species: T. aquaticus leuB (Kirino, H. &Oshima, T., 1991, J. Biochem. 109, 852-857) (bottom), T. thermophilusleuB (Croft, J. E., Love, D. R. & Bergquist, P. L., 1987, Mol. Gen.Genet. 210, 490-497) (middle) and T. flavus leuB (top, this study). Thebar underneath the T. flavus restriction map is the smallest subclone ofthe DNA fragment that was able to complement the leuB mutation in E.coli KC8.

FIG. 5 shows PCR primer sequences for amplification of the kan andkan^(tr2) genes. Primer sequences are shown in bold type where theycorrectly pair with the template sequence. Top line, coding sequence forthe kan gene; bottom line, anticoding strand of the kan gene. Arrowsindicate direction of primer elongation. "PCR 3' primer (containing aNcoI site) is shown (SEQ ID NO: 4). PCR 5' primer (containing a NcoIMscI and DraI site), is shown (SEQ ID NO:3). Part of sense strand of thekan gene (SEQ ID NO: 5) and (SEQ ID NO: 7). Part of anti-sense strand ofthe kan gene (SEQ ID NO: 8) and (SEQ ID NO:6)."

FIG. 6A-6B show Southern hybridization of T. flavus total DNA showinggene replacement of kan^(tr2) into the leuB region of the chromosome.(A) NcoI and BamHI-double-digested DNA (lanes 1-8); DNA from Kan^(R)Leu⁻ transformants, lanes 4-6. DNA from untransformed cells, lanes 3, 7,8. DNA size markers (kb units) indicated to the right. (B) Schematicrepresentation of DNA from pTG100kan^(tr2) transformants showing theinsertion of the kan^(tr2) gene (black arrow) into the leuB gene (shadedarrow) of the T. flavus chromosome (shaded line). Striped bar indicatesDNA fragment used as the probe in the hybridization reaction.

SUMMARY OF THE INVENTION

The instant invention is directed to recombinant DNA which contain a DNAfragment isolated from a Thermus strain, such as Thermus flavus, whichcontain a site for insertion of a coding sequence for a heterologousprotein, and which contain a coding sequence which directs the insertionof the DNA fragment into a regulated region of a Thermus chromosome sothat the expression of the exogenous protein is regulated by the Thermuschromosome. In a prefered embodiment the Thermus strain is Thermusflavus.

In one embodiment, a recombinant DNA as above, preferably contains a 3kb DNA fragment isolated from Thermus which complements a leuB mutationin E coli MC1066, or equivalents thereof.

In another embodiment, a recombinant DNA as above, preferably contains a1.7 kb fragment isolated from Thermus which complements a pyrE mutationin E. coli BW322, or equivalents thereof.

In a preferred embodiment, a recombinant DNA as above, contains a DNAfragment corresponding to the leuB gene locus isolated from Thermus thatcontains an exogenous DNA insert which is capable of being expressedunder the control of the leuB regulatory signals.

The instant invention and the compositions which flow naturally from theteachings of the instant invention encompass methods of inserting anexogenous DNA sequence into a targeted chromosomal DNA region of athermophilic microorganism comprising the steps of; constructing aplasmid vector which contains a targeting DNA sequence which correspondsto the targeted chromosomal DNA, inserting into the plasmid vectortargeting DNA sequence an exogenous DNA, the insertion of whichinterferes with the normal expression of the targeting DNA sequence,transforming a host cell with the plasmid vector, selecting stabletransformants by screening at temperatures above 55° C., confirminggene-replacements by southern blotting.

The instant invention also contemplates the above method where theinsertion does not result from a interruption of the gene function. Alsoencompassed are fusion proteins.

Thus the instant invention also encompasses a host cell which has beentransformed by the methods of the instant invention. Specific strainswhich have been transformed by the methods of the instant inventioninclude T. flavus AT62.

In a particular embodiment, the methods of the instant inventionencompass where the exogenous DNA sequence encodes the kan gene.

Further, the methods of the instant invention encompass where thetargeting DNA sequence and the targeted chromosomal DNA sequence is theleuB gene.

The instant invention also encompasses methods of evolving thermostableproteins which comprises using cells transformed by the methods andcompositions of the instant invention, and subjecting them to elevatedtemperatures for selection of mutated thermostable proteins. In aprefered embodiment, the compositions are stable cells which have beentransformed by the methods of the instant invention, to express aheterologous protein.

Thus the instant invention embodies a recombinant DNA which contains aDNA fragment isolated from Thermus, which DNA fragment contains a sitefor insertion of an exogenous DNA coding sequence, and which DNAfragment contains a DNA coding sequence which directs the targetedinsertion of the DNA fragment into a region of a Thermus chromosome,such that the exogenous protein is expressable.

The instant invention further provides for a recombinant DNA as abovewhich contains a DNA fragment isolated from Thermus flavus, whichcomplements a leuB mutation in E. coli MC1066. In the preferedembodiment the instant invention provides for a recombinant DNA as abovewhich contains a 3 kb DNA fragment isolated from Thermus flavus whichcomplements a leuB mutation in E. coli MC1066.

Further the instant invention embodies a recombinant DNA which containsa DNA fragment isolated from Thermus flavus which complements the pyrEgene, which is capable of being expressed under the control of Thermusregulatory signals.

The instant invention also embodies a recombinant DNA which contains aDNA fragment corresponding to the leuB gene isolated from Thermus thatcontains an exogenous DNA insert which is capable of being expressedunder the control of leuB regulatory signals.

In addition the instant invention embodies a recombinant DNA whichcontains a DNA fragment corresponding to the pyrE gene isolated fromThermus that contains an exogenous DNA insert which is capable of beingexpressed under the control of pyrE regulatory signals.

The instant invention also embodies the regulation of expression byother signals.

The instant invention provides methods of isolating proteins comprisinggrowing cells in media and isolating protein, wherein said cells weretransformed with a vector, by the insertion of a targeting DNA sequencefrom said vector into a corresponding targeted chromosomal DNA region ofa thermophilic microorganism, wherein said targeting DNA sequencecontains an exogenous DNA sequence, where the insertion of the exogenousDNA sequence interferes with the normal expression of the targeting DNAsequence, and said exogenous DNA sequence encodes for the desiredprotein, where the expression of the protein is under the regulation ofa chromosome.

In preferred embodiments this method is used where the exogenous DNAsequence encodes the kan gene, where the targeting DNA sequence and thetargeted chromosomal DNA sequence is the leuB gene, and where thetargeting DNA sequence and the targeted chromosomal DNA sequence is thepyrE gene.

The instant invention also encompasses methods of producing a stablehost cell transformant which produces a protein comprising, growing hostcells and selecting for a stable host cell transformant where the hostcells have been transformed with a vector, where said vector transformsthe host cell by inserting a targeting DNA sequence into a targetedchromosomal DNA region of a thermophilic microorganism, which targetingDNA sequence contains an exogenous DNA sequence, which exogenous DNAsequence interferes with the normal expression of the targeting DNAsequence, and the expression of the exogenous DNA sequence is regulatedby the chromosome.

In preferred embodiments this method is used where the exogenous DNAsequence encodes the kan gene, where the targeting DNA sequence and thetargeted chromosomal DNA sequence is the leuB gene, where the targetingDNA sequence and the targeted chromosomal DNA sequence is the pyrE gene.

Thus the instant invention also provides for methods of evolvingthermostable proteins which comprises subjecting host cells to elevatedtemperatures for selection of mutated thermostable proteins in astep-wise fashion, where said host cells were produced by, growing hostcells and selecting for a stable host cell transformant, where the hostcells have been transformed with a vector, where said vector transformsthe host cell by inserting a targeting DNA sequence into a targetedchromosomal DNA region of a thermophilic microorganism, which targetingDNA sequence contains an exogenous DNA sequence, which exogenous DNAsequence interferes with the normal expression of the targeting DNAsequence, and the expression of the exogenous DNA sequence is regulatedby the chromosome.

In preferred embodiments, this is done where the exogenous DNA sequenceencodes the kan gene, where the targeting DNA sequence and the targetedchromosomal DNA sequence is the leuB gene, and where the targeting DNAsequence and the targeted chromosomal DNA sequence is the pyrE gene.

The instant invention also further provides for a host cell which hasbeen produced by the above methods, and protein which has been isolatedand/or produced by the above methods.

In one embodiment the invention encompasses a cell which is a strain ofThermus flavus, which has been transformed by plasmid vector pTG101.

In one embodiment the instant invention encompasses a nucleic acidsequence comprising the sequence which encodes the Thermus flavus pyrEgene (SEQ ID. 1). Which is useful as an additional target for specificchromosomal integration in Thermus. Further the instant inventionprovides for the protein translated from the pyrE gene which is apolypeptide comprising the amino acid sequence of pyrE (SEQ. ID. 2) andis a thermostable dehydrogenase and is useful for syntheticapplications.

Other embodiments of the methods and compositions of the instantinvention will be readily apparent to one of ordinary skill in the artfrom the teachings of the instant disclosure, which would allow such aperson to practice the methods of the instant invention.

DETAILED DESCRIPTION OF THE INVENTION

The presently available thermogenetic systems offer a range oftemperatures at which selection may be carried out. While thesetemperatures were reasonably interesting for academic work, they werenot sufficient for the system to be useful to thermostabilize enzymesand proteins at the levels needed in commercial applications. There wasa limitation to the process because of the upper growth temperature ofthe host organism. An improved thermogenetic system would allow forselection at higher temperatures and a wider range of temperatures toprovide solutions to the limitations of the previous efforts.

To circumvent the limitation of temperature, we chose to work with amore extreme thermophile, Thermus flavus, which has an upper growthlimit, at least 10° C. higher than B. stearothermophilus.

Starting with the double thermo-stabilized kan^(tr2) gene (provided byH. Liao) we teach a gene transfer system using chromosomal integration.In our system, however, we inserted kan^(tr2) into the Thermus flavusleuB gene rather than into the slpA gene of Thermus thermophilus (Lasaet al., 1992). LeuB has been well-characterized in related organisms, itwas also predicted to have a benign mutant phenotype. Since kan^(tr2) isselectable when inserted into a common metabolic gene like leuB and at arelatively low expression level, it is possible that it will also beselectable in many other sites as well. This opens the possibility ofusing kan^(tr2) for mutational analysis of the entire Thermuschromosome. We have also cloned two other metabolic genes of Thermusflavus, pyrE and his, which will be useful markers in chromosomalinsertion systems. Thus the instant invention encompasses the use of themethods of the instant invention for the directed insertion of DNAsequences into other metabolic genes of T. flavus.

The instant invention circumvents the problems encountered by othergroups and provides a host-vector system that would lend itself tofurther thermogenetic selections. The instant invention demonstratesthat a low- or regulatable-expression system has significant advantagesover the high expression system.

The teachings of the instant specification provide a new integrativegene transfer system based on the leuB gene insertion site, and teachhow this system enables one to perform further thermo-genetic selectionson the kan^(tr2) gene. Surprisingly, due to the unique properties of oursystem, the observed potential for thermo-genetic selections with ournew system is significantly greater than the additional 10° C. expectedfrom the new host.

We have been able to obtain mutants in kan^(tr2) that were able to growat 65° C. having varying degrees of linkage between Leu⁻ and thekan^(tr2+) phenotypes. The differences in linkage indicated thatdifferent types of mutations were probably obtained. Mutations showing ahigh linkage would have the greatest probability of being truethermo-stabilizing mutations in kan^(tr2) ; low linkage could indicatemutations in the leuB regulatory region (high-expression mutants) or inother genes controlling the level of kanamycin in the cell.

The instant specification teaches a new integrative gene transfer systemin the extreme thermophile Thermus flavus using kan^(tr2) as theselectable marker and leuB as the site of insertion. This system isuseful for performing thermo-genetic selections on enzymes and proteinsof industrial importance beyond the limitations of any other currentlyavailable system.

Using the kan^(tr2) allele, the Examples of the instant specificationdemonstrate how changing the host-vector environment from a moderatethermophile to an extreme thermophile can be used to allow for furtherthermo-genetic selections. Using the leuB insertion site, the methods ofthe instant invention were able to produce an additional 25° C. oftemperature-shifting potential for further thermo-genetic selections onthe kan^(tr2) allele.

Thus the instant specification teaches the recognition of the importanceof the choice of host-vector in determining thermo-genetic potential ofa system. Using slpA as the insertion site, for example, kan^(tr2) canbe selected at 70° C.; using leuB as the insertion site, however, thesame allele was selectable only up to 55° C. Since the power ofthermo-genetics is in large part determined by the potential fortemperature-upshifts, the leuB environment of the instant inventionprovides a much greater range (about 55° C.-85° C. as opposed to about70° C.-85° C.).

The disadvantage of a presumably lower expression system like leuB isseen when a mesophilic gene is used, such as kan. We observed, forexample, that the wild-type kan could not be used as selectable markerin leuB even at the relatively low selection temperature of 45° C. Themethods of the instant invention teach that in order to overcome thelimitations of either a single high-expressing system or a singlelow-expressing system, that they both be used at the appropriate pointsin the process of making thermostable proteins. Ultimately, the mostconvenient and useful thermogenetic system is one that allows forregulated expression of the gene of interest. A simple change ofconditions (for example, in nutrient supplementation) could be used toraise or lower expression levels and allow for a sophisticated controlover the appropriate selection temperature for thermo-stabilizingmutations.

The T. flavus-leuB system of the instant invention is useful for thefurther thermo-stabilization of kan^(tr2). It is superior to the otherchromosome-integration system using a slpA gene because mutations inleuB are not harmful to the host as are slpA mutations, and because ofthe wider range available for thermo-genetic selections presumablybecause of the lower expression level of the leuB gene. The instantmethods and compositions demonstrated in the instant disclosure areuseful and convenient because plasmid constructions can be made in E.coli, and gene replacements can be constructed efficiently in Thermus,and the leuB region offers the possibility for regulated control overthe expression level of the cloned gene of interest for thermogeneticselections. The constructs of the instant invention are useful asvectors for inserting and expressing exogenous genes in an extremethermophile.

Other thermostability approaches have used plasmid-based systems withoutchromosomal integration. This is because plasmids are generally thoughtof as being stable, easy to work with, easy to handle, easilytransferred from one strain to another, and easy to make large amountsof DNA from. Unfortunately, in Thermus, plasmid based systems which havebeen tried have yielded inconsistent results. Thermus plasmids areunstable, tend to form multimers. Most researchers who have triedThermus have reverted back to Bacillus for dependability of working withthe plasmids, although they have lost the advantages of the highertemperature range. A major advantage of the Thermus organisms overBacillus is that a range of Thermus organisms exist with differentoptimal growth temperatures, and that proteins native to Thermus appearmore stable outside the cytoplasm. The stability in Thermus appears tolie in the proteins themselves, whereas in Bacillus there appears to bea cytoplasmic factor involved. Thus the instant invention provides foran effective means of using Thermus by teaching methods for stabletargeted chromosomal integration for protein expression under specificregulation.

In fact, in the only other reports of inserting exogenous genes intoThermus have been directed not at stable gene transfer, but atinsertional mutagenesis techniques for inactivating specific genes, orin trying to utilize a transposon in Thermus aquaticus. Thermusaquaticus, however has a lower temperature range and is not easilytranformed by exogenous DNA. Lasa et al inserted the kan^(tr2) gene ahighly expressed region of the chromosome to attempt disruption.Unfortunately this cause a debilitating phenotype making it a badgeneral integration system.

Very few workers have realized the significant advantages of T. flavus.T. flavus is closely related to T. thermophilus but, in our hands, has atransformation efficiently nearly 10× that of T. thermophilus. In fact,it was surprising to find that over 1% of all DNA molecules incubatedwith competent T. flavus cells are recombined into the chromosome of T.flavus in a transformation. This is important for transforming a seriesof mutagenized DNA molecules into the chromosome to increase the numberof mutants which can be screened. Our system also has a lower expressionlevel which is regulated by the leucine biosynthetic operon of T.flavus. This is very important for adjusting expression levels prior tothe instant invention all workers in the field have focus on highprotein expression at high temperatures in the belief that this was themost effective way to measure and select for mutations cenferringthermostability.

Our system also incorporates the use of kan^(tr2) at 55° C. instead of65° C. which allows us to have a broader temperature range for selectionof thermostabilizing mutations. The instant invention allows us togenerate thermostable mutations in a stepwise fashion. For example, bystarting the mutation process at 55° C. instead of 65° C., we have alarger range of temperatures in which mutations can occur before wereach the upper growth limit of the organism.

The advantages of regulation and control over expression and expressionlevels are that the perceived thermosability of an enzyme is related toits expression level as well as the temperature.

In an effort to increase the thermostability of a protein, theselectable or screenable level of thermostability is observed as afunction of how much protein is present at a given temperature. Themeasurable thermostability in a genetic selection or screen is dependanton both the half-life of the protein and the expression levels of thegene. In an overexpressing system, the temperature range available toincrease thermostability is reduced since more total activity is presentat any given time. While the In a low expression system, there is muchless protein at any given temperature giving a wider range oftemperatures available. Thus the instant invention provides for a moreefficient and flexible selection of thermostable proteins over a widerrange of temperatures.

The level of expression of the gene of interest is very important forthe practical application of a thermostabilization process. For proteinswhich are less stable to begin with, a higher expression level isinitially desired. Once thermostabilizing mutations have been generatedto allow the protein to function near the upper growth limit of the hostorganism, a lower expression level is desired. This will effectivelyreduce the amount of protein in the cell and therefore reduce thetemperature which the protein is effective at. The process can onceagain be repeated to the upper growth level of the organism.

Further, by allowing for regulation, lethal mutations can be controlledand growth of the organism continued under non-lethal conditions.

The methods and compositions of the instant specification also providefor the pyrE nucleic acid sequence from Thermus flavus which is usefulas a selectable target stable chromosomal integration into Thermus. Thusthe instant invention provides for the stable integration into thechromosome of Thermus at two sites with the same or different proteins.

Thus the instant invention provides methods and compositions which willallow for the insertion and expression of a exogenous protein underspecific regulation in Thermus where such protein can be subjected tofurther methods of the instant invention to generate more thermostablemutations.

The Examples described below are only intended by way of illustration,of the embodiments of the instant invention, and are in no way limitingas to the scope of the instant invention. One with ordinary skill in theart would be able to use the teachings of the instant specification toformulate and practice modifications and substitutions which are wellwithin the scope of the instant disclosure.

EXAMPLE 1

Propagation of bacteria

Strains and Plasmids. Thermus flavus (T. flavus) AT62 is available fromthe American Type Culture Collection, #33923 (Saiki, et al, 1972, Agric.Biol. Chem. 36, 2357-2366). Thermus thermophilus (T. thermophilus) HB8is available from the American Type Culture Collection, #27634, (Oshima,1974, International Journal of Systematic Bacteriology 24:102-112).Thermus aquaticus (T. aquaticus) YT1 is available from the American TypeCulture Collection, #25104, (Brock & Freeze, 1969, J Bacteriol98:289-297). E. coli KC8 F⁻ hsdR³¹ X1488, leuB6, D(lac)X74, galE15,galK16, trpC9830, pyrF74::Tn5(Km^(R)), hisB463, rpsL(Str^(r)) wasobtained from K. Struhl, Harvard University. E. coli MC1061 (Casadaban,et al., 1980, J Bacteriol 143:971-980) F⁻ araD139 D(ara-leu)7696 galE15galK16 D(lac)X74 rpsL (Str^(r)) hsdR2 (r_(K) ⁻ m_(K) ⁺) mcrA mcrB1; andE. coli MC1066 (Casadaban, et al., 1983, Recombinant DNA. Methods inEnzymology 293-308) F⁻ hsdR⁻ X1488, leuB6, D(lac)X74, galE15, galK16,relA1, spoT1, trpC9830, pyrF74::Tn5(Km^(r)), rpsL150(^(r)) are in theThermoGen strain collection. E. coli BW322, Hfr (PO45 of Hfr KL 16, serA(63'), lysA (61')), λ⁻, relA, rfa-210::Tn10,pyrE70, spoT1, thi-1, wasobtained from B. Bachmann (E. coli Genetic Stock Center), E. coli DH5a,F⁻ f80dlacZDM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(r_(k) ⁻ m_(k) ⁺)deoR thi-1 supE44 λ⁻ gyrA96 relA1 was obtained from (Gibco/BRL LifeTechnologies). Plasmid pUB110 was obtained from N. Welker, NorthwesternUniversity; (Bacillus Genetic Stock Center, Ohio State University,Columbus, Ohio). Plasmid pUC-TK101 carrying the doubly thermo-stabilizedkan gene (Y80/K130 (Liao, 1986; Matsumura, 1986) was obtained from H.Liao, University of Wisconsin. E. coli plasmids used included pUC19(Sambrook, et al., 1989), pBluescript_(II) -SK(+) (Stratagene), and theLorist 6 cosmid vector (Gibson, et al., 1987., Gene 53:275-281).

General growth media and conditions. Complex broth medium for growth ofall Thermus strains was described in the American Type CultureCollection catalog as Medium 697, Thermus medium (TT); TT agar mediumadditionally contained 15 g/L Bacto-Agar (Difco). Transformation media(TM) for T. flavus was described by Koyama et al. (Koyama, et al., 1986,J Bacteriol 166:338-340). The minimal agar medium (Tmin) previouslydescribed by Yeh and Trela (Yeh and Trela, 1976, J Biol Chem251:3134-3139) was used with the addition of 0.1% Casaminoacids (unlessotherwise noted) for the phenotypic analysis of T. flavus auxotrophs.The composition of Tmin is as follows: sodium glutamate, 3 g; Thermusbasal salts (5000×), 0.2 ml; after autoclave sterilization, 0.1% biotin,100 ml; 1% thiamin, 10 ml; and 0.1% nicotinic acid, 50 ml, were added.Thermus basal salts (5000×) were prepared as follows: nitrilotraceticacid, 0.5 g; CaSO₄.2H₂ O, 0.3 g; MgSO₄.7H₂ O, 0.5 g; NaCl, 0.04 g; KNO₃,0.515 g; NaNO₃, 3.45 g; Na₂ HPO₄, 0.55 g; FeCl₃, 0.014 g; MnSO₄.H₂ O,0.11 g; ZnSO₄ 7H₂ O, 0.025 g; H₃ BO₃ 0.025 g; CuSO₄, 0.008 g; Na₂MoO₄.2H₂ O, 0.00125; CoCl₂.6H₂ O, 0.0023 g; EDTA, 0.769 g; dissolved indistilled water, 1 liter. Standard growth conditions for T. flavus brothcultures were shaking at 280 rpm, one-inch circular displacement and 65°C. Agar cultures were incubated under water-saturated atmosphericconditions also at 65° C. unless otherwise stated. M9 minimal media, M63minimal media, YT, and LB media (Sambrook, et al., 1989) supplementedwith appropriate nutrients and antibiotics were used for general growthof E. coli strains.

EXAMPLE 2

Isolation of Ura⁻ Thermus and 5FOA selection

Construction of Ura⁻ mutants of Thermus. Mutants in the uracilbiosynthetic pathway (Ura⁻) are extremely useful for genetic studies andgenetic manipulation of bacterial and fungal organisms since a positiveselection exists for either the loss or the presence of gene function.In yeast and E. coli, resistance to 5-Fluoro-orotic acid (5FOA) can beused as a positive selection (Boecke, et al, 1984. Mol. Gen Genet.197:345-346) for mutants in several genes including two in the uracilbiosynthesis pathway:URA1 and pyrE (coding for orotate phosphoribosyltransferase, OPRT) and URA3 and pyrF (coding for orotidine-5'-phosphatedecarboxylase). By plating onto minimal media lacking uracil, onlyorganisms which have wild-type pyr genes will be able to grow, allowingfor a positive selection for non-mutant strains.

In order to obtain Ura⁻ strains of Thermus, mutagenesis of severalThermus strains including T. thermophilus, T. aquaticus, and T. flavuswas performed by one of two methods. In the first method, we plated foruv-induced mutations by irradiating cells with 305 nm ultraviolet lightfor periods of time between two seconds and two minutes. Cells wereplated onto TT media and exposed face-down, without the lid, on a uvtransilluminator. An optimal mutagenesis by uv treatment was determinedby selecting time points which killed between 20% and 50% of the cells.For all strains, this was approximately 20 seconds. Plates were allowedto grow for one to two days. Two ml of TT broth was added to the plates,mixed with the cells, and the mixture was scooped up for re-plating.

Optimal concentration of 5FOA (obtained from Sigma Chemicals) wasdetermined by plating overnight cultures of the various wild-typeThermus strains onto a series of dilutions of 5FOA ranging from 20 μg/mlto 1 mg/ml and observing the minimal concentration of 5FOA needed toreduce background growth of bacteria on the media. The selection wasfound to work well with concentrations of 5FOA above 300 μg/ml for T.aquaticus and T. thermophilus, and above 500 μg/ml for Thermus flavus.Once mutants were generated they could be streaked out on mediacontaining lower concentrations of 5FOA (150-300 μg/ml) since 5FOA wasvery expensive.

The pooled mutant mixtures plated onto TT media containing theappropriate concentration of 5FOA. Colonies which arose on the mediawere restreaked onto TT media containing 5FOA. Mutants could alsosimilarly be generated by plating directly onto 5FOA selectiveconcentrations and performing uv mutagenesis on these plates.

A second method which was developed and used to select for spontaneouslygenerated 5FOA resistant mutants. The method consisted of plating theThermus strains onto TT media, punching a hole in the center of theplate made aseptically with the back-end of a sterilized pasteurpipette, adding 500 μl of a 1 mg/ml solution of 5FOA to the holeallowing the 5FOA to diffuse out, and incubating at 65° C. Mutantsresistant to 5FOA appeared after two days and were selected from theedges of a ring of killing created by the diffusing 5FOA. Mutants werepurified by restreaking onto media containing 150-300 μg/ml of 5FOA.

Confirming Ura mutations. To verify that the mutants obtained were inthe uracil biosynthetic pathway, the mutant colonies were patched ontoTmin media supplemented with uracil (50 μg/ml), onto Tmin withouturacil, and onto TT media containing 150-300 μg/ml of 5FOA. The mutantswhich grew on Tmin supplemented with uracil and on TT with 5FOA but noton Tmin without uracil were identified and further characterized.

Reversion testing. The reversion frequencies of the mutations wereanalyzed by growing overnight cultures of the mutants and platingdilutions onto either TT rich media or Tmin without uracil added. Thereversion frequencies of the mutations were calculated by dividing thetotal number of colony forming units per ml obtained on minimal mediawith the total number of colony forming units per ml on rich media. Formost of the colonies, the reversion averaged about 5×10⁻⁶ to 5×10⁻⁷which is consistent with a standard bacterial spontaneous mutationfrequency. One in particular, TGF35, was used for the experimentsdescribed here which had a reversion frequency of about 1×10⁻⁷.

EXAMPLE 3

Cloning of pyrE

Isolation of Thermus DNA. Genomic DNA of T. flavus was prepared bytreatment of overnight Thermus cultures with 2% SDS in 50 mM Glucose, 25mM Tris.HCl, pH 8 and 30 mM EDTA at 42° C. for 5 minutes and another 10minutes in the presence of Proteinase K (20 mg/ml), followed by a 1 hPhenol/Chloroform/Isoamylalcohol extraction (25:25:1) and precipitationwith 1.5 volumes of 100% ethanol. Mini-scale DNA preparations forlibrary screening were carried out in 96-well microtiter plates.Large-scale and small-scale DNA preparations were performed usingstandard procedures described in (Sambrook, et al., 1989).

Construction of gene library. A λ-ori cosmid, Lorist 6 was used asvector and the E. coli strain DH5α as host for the construction of thegene library. The cloning of size-fractionated, Sau3A-digested DNA of T.flavus was achieved using HindIII-BamHI and EcoRV-BamHI "arms" of thecosmid (Sambrook, et al., 1989). Size fractionation was performed byultracentrifugation through 10-40% sucrose gradients and fractions wereanalyzed by conventional electrophoresis. Fractions with averagefragment sizes of 35-45 kb were used for cloning. Purified insert (300ng DNA) was dephosphorylated to avoid cloning of non-neighboring DNAfragments, and ligated with an equimolar amount of cosmid arms in 10 mlof ligation mixtures and packaged in vitro into I heads. After infectionof E. coli DH5α and plating on LB medium containing 30 μg/ml ofkanamycin, a total of 700 cosmid clones were recovered. Individualcolonies were picked and grown in 96-well microtiter plates and storedin 40% glycerol at -70° C.

Screening for Ura⁻ containing region by transformation into Thermus.Since thermophile genes do not always complement E. coli mutations,because of either low expression or low activity in E. coli at 37° C.,we decided to screen the T. flavus gene library in TGF35, alow-reversion Ura⁻ strain of Thermus flavus obtained by screening on5-fluoro-orotic acid (5FOA). Individual recombinant gene library cloneswere grown in 96-well microtiter plates and mini-scale cosmid DNApreparations were performed. This DNA was transferred onto a lawn ofcompetent T. flavus TGF35 cells on a Tmin media lacking uracil byspotting approximately 2 μl of DNA using a 48-prong replica tool(corresponding to 1/2 of a microtiter plate). A total of 672 recombinantcosmids were screened for their ability to transform the TGF35 toprototrophy. Cosmids containing a non-mutant sequence which covered ahomologous region to the ura⁻ mutation in TGF35 were able to recombineinto the Thermus chromosome by homologous recombination, replacing themutant phenotype with a wild-type one and allowing growth on the minimalmedia. Positive results scored as growth on the minimal media and wereseen with 28 cosmids. Cosmid 2A6 was chosen for furthercharacterization.

Subcloning of the pyrE gene. Since TGF35 was an uncharacterized TF ura⁻mutant we tested the cosmid clones for the ability to complement both E.coli pyrE (BW322) and pyrF (MC1066) mutants. Cosmid 2A6 was partiallydigested with Sau3A, dephosphorylated to avoid ligation ofnon-neighboring pieces and subcloned into the BamHI site of pUC19. Theligation mixture was used to test transformation of E. coli BW322 (pyre)and MC1066 (pyrF) to prototrophy. Only BW322 was able to be transformedby selection on minimal media lacking uracil indicating that the cloneswere pyrE clones. Small-scale plasmid DNA preparations from 40individually picked clones revealed the minimal insert size of 4 kb inplasmid pVUF10. A physical map of pVUF10.5 is shown in FIG. 1. Thesmallest Thermus DNA piece able to complement the E. coli mutant wasfound to be the 900 bp insert of pVUF10.5.9.

Determination and analysis of the nucleotide sequence of the pyrE gene.The 1.7 kb KpnI fragment of subclone pVUF10.5 was cloned in differentorientations into pBL2-SK(+) and designated as pTG-F or pTG-R.Undirectional deletions were generated from pTG-F and pTG-R usingExonuclease III and S1 nuclease. We have sequenced the DNA clone with ³⁵S corresponding to the KpnI fragment using the dideoxy sequencing methodof Sanger. The sequence presented in FIG. 2 contains one major openreading frame (ORF) starting with ATG (position 262) and ending with TAG(position 810). The open reading frame shows homology with other genesencoding OPRT including E. coli and Bacillus subtilis.

EXAMPLE 4

Cloning of leuB

Cloning of leuB from T. flavus. Plasmid pTG100 (FIG. 3A), carrying theT. flavus leuB gene, was isolated by complementation of the leuB6mutation in E. coli KC8. It was selected from a library of plasmidscreated through the ligation of T. flavus DNA, partially digested withSau3A, to the BamHI site of the vector pTZ18R (Pharmacia P-LBiochemicals Inc., Milwaukee, Wis.).

Plasmid pTZ18R (Pharmacia P-L Biochemicals Inc., Milwaukee, Wis.)linearized with BamHI was mixed under ligation conditions with 2-3 kbSau3A fragments of T. flavus total DNA. The ligation products were addedto competent E. coli KC8 cells, and the cells were plated on M9 minimalmedium (Sambrook, et al., 1989) containing histidine (50 μg/ml) andtryptophan (50 μg/ml), uracil (10 μg/ml), ampicillin (100 μg/ml), andlacking leucine for the selection of Leu⁺ transformants. The Leu⁺ cloneswere analyzed and the plasmid with the smallest insert was designatedpTG100 (FIG. 3A).

The 3 kb clone in pTG100 is analogous in its restriction map to the leuBgenes from T. thermophilus (Nagahari, et al., 1980, Gene 10:137-145;Tanaka, et al., 1981, J Biochem 89:677-682; Croft, et al., 1987, Mol GenGenet 210:490-497) and T. aquaticus (Kirino and Oshima, 1991, J Biochem109:852-857) which encode the enzyme 3-isopropylmalate dehydrogenase(FIG. 2). This probably reflects a high degree of sequence homologybetween the three species since the leuB genes of T. thermophilus and T.aquaticus are already known have 87% homology in nucleotide sequence(Kirino, H. & Oshima, T., 1991, J. Biochem. 109, 852-857). Theorientation and location for T. flavus leuB within pTG100 is based onthis comparative analysis, specifically because the sites for BamHI,XhoI, NcoI, MscI, and XmnI all appear in the same order and relativespacing as the T. thermophilus sequence. This map indicates that theinsert in pTG100 contains the entire leuB open reading frame,approximately 200 bp of upstream sequences, and 1.5 kb of downstreamsequences. The smallest fragment containing the entire proposed leuBgene is the 1.1 kb BamHI fragment (FIG. 4); which was subcloned into theBamHI site of pUC19 and found to complement leuB6 in E. coli MC1066.

EXAMPLE 5

Inserting kan into leuB

Insertion of kan and kan^(tr2) into leuB. The kan gene from pUB110(Lacey and Chopra, 1974, J Med Microbiol 7:285-297; Matsumura, et al.,1984, J Bacteriol 160:413-420) and the kan^(tr2) gene from pUCTK101(provided by H. Liao) were inserted into the unique NcoI site of leuB inpTG100 to create pTG100kan and pTG100kan^(tr2) (FIG. 3B), respectively.

The polymerase chain reaction was used to produce the two kan genes withNcoI sites at each end (FIG. 5). We used the PCR method described byPonce and Micol with modifications, using buffer III and 30 cyclesinstead of their recommended 10 cycles to generate our PCR products(Ponce, M. R. & Micol, J. L., 1991, Nucl. Acids Res. 20, 623).Oligonucleotide primers were obtained from Operon Technologies, Inc.(Molene, Calif.).

Circular pTG100 DNA was mixed with a PCR DNA in the original PCRreaction buffer containing TaqI polymerase. NcoI was added with therecommended reaction buffer from NEB, and the reaction was allowed toincubate at 37° C. After 3h NcoI was inactivated by heating to 65° C.for 20 min and allowed to cool slowly to room temperature, during whichtime the TaqI polymerase was able to fill-in the NcoI overhangs tocreate blunted ends on both the PCR DNA and pTG100 (FIG. 4). The DNA wasethanol precipitated and then resuspended in a solution suitable forligation of the DNA in the reaction. The products of ligation weretransformed into E. coli MC1061 and transformants were selected on YTagar plates ("Molecular Cloning." 2nd Ed. Sambrook, Fritsch and Maniatiseds. 1989 Cold Spring Harbor Laboratory Press) supplemented with 10 or20 μg/ml of kanamycin and 100 μg/ml of ampicillin. Five transformantsfrom each ligation were analyzed: 9 of which carried a single insert inthe same orientation as the leuB gene in pTG100. The tenth transformant,a kan double clone, carried both inserts in the opposite orientation toleuB. The insertion of the kan gene into the NcoI site of pTG100 wasconfirmed by restriction analysis of the plasmids from transformantsthat were resistant to both Ap (100 μg/ml) and Km (10 μg/ml).Restriction analysis showed that the NcoI sites had been converted, aspredicted, through the joining of blunt NcoI sites to produce NsiI sitesat the junctions (FIG. 5).

Alternatively, the kan^(tr2) gene could be cloned into the TthIIII siteto disrupt the pyrE gene using the same methods as above.

EXAMPLE 6

Transformation of T. flavus and Confirming insertion of exogenous gene

Testing for disruption of the leuB gene. To test for the Leu⁺complementation phenotype, pTG 100 plasmid DNA, plasmid DNA from one ofthe kan transformants (pTG100kan) and one of the kan^(tr2) transformants(pTG100kan^(tr2)), was used to transform E. coli MC1066. As expected,only pTG100 plasmid DNA was able to complement the leuB6 mutation,indicating that the insertions of kan and kan^(tr2) had inactivatedleuB. Finally, we observed that the expected NcoI sites were not presentat the ends of kan and kan^(tr2) in their respective plasmids, but werereplaced by NsiI sites. Apparently, the NcoI ends were filled-in to formblunt ends by Taq polymerase, which had not been removed from thereaction, and upon ligation with the similarly filled-in vector ends,created the NsiI sites. Three other clones which were isolated in asimilar experiment which contained an Nco I site upstream(pTG100kan^(tr2) cs), downstream (pTG100kan^(tr2) sc), or at both ends(pTG100kan^(tr2) cc) of the kan^(tr2) gene insertion with an NsiI siteat the other position.

Disruption of chromosomal leuB by gene-replacement with kan^(tr2) usingpTG100kan^(tr2). Both pTG100kan and pTG100kan^(tr2) were used intransformation reactions with T. flavus. A transformation protocol forT. flavus AT62 (ATCC 33923) has been described (Koyama, et al., 1986, J.Bacteriol. 166:338-340). The procedure used in this study for the wildtype strain was a slight modification of Koyama's. Individual coloniesfrom TT plates were used to inoculate 2 ml test tube cultures of TM andgrown overnight (17 h) at 65° C., 280 rpm. 1.6 ml of the test tubeculture was used to inoculate 50 ml of TM broth in a 250 ml Ehrlenmeyerflask and the flask was incubated at 65° C., 280 rpm for 4 h.Transformations involved adding 1 μg of plasmid or 4 μg of chromosomalDNA to 500 μl portions of the 4 h culture; the cells and DNA wereincubated at 65° C. while shaking, for 1 h. The cells and DNA were thendiluted and plated on TT plates containing the appropriate supplementskanamycin sulfate (Sigma Chemical) was used at 20 μg/ml final agarconcentration! and incubated at 65° C. for 2-3 days until transformantsarose. The growth temperature and incubation temperature fortransformation plates was 65° C. instead of at 70° C. as described byKoyama (1986).

Selections for transformants were performed on agar containing 20 μg/mlof kanamycin. The initial selection temperature was chosen to be 65° C.as described in the original B. stearothermophilus system (Liao, 1986)and in later systems developed for Thermus (Mather and Fee, 1992, ApplEnviron Microbiol 58:421-425; Lasa, et al., 1992, J Bacteriol174:6424-6431; Lasa, et al., 1992, Molec Microbiol 6:1555-1564).Analysis of the colonies appearing at this temperature revealed that themajority (averaging 82%) of the pTG100kan^(tr2) transformants were notstably kanamycin-resistant or Leu⁻. The remainder of the pTG100kan^(tr2)transformants were generally larger colonies which were Leu⁻. Thesecolonies restreaked at 65° C. on kanamycin media, although grew poorly.None of the transformants from the pTG100kan plasmid containing thenon-thermostable kan gene were stably kanamycin-resistant or Leu⁻. Insummary, although the Leu⁻ kanamycin resistant transformants could beisolated at 65° C., they were difficult to find among the more abundantbackground growth. The pTG100kan transformations produced no stablekanamycin-resistant or Leu⁻ colonies.

We next performed Southern analyses on a sample of Leu⁺ and Leu⁻colonies from the pTG100kan^(tr2) transformations. Chromosomal DNA fromthe following: three pTG100kan^(tr2) Leu⁻ transformants, two Leu⁺colonies, and the parental wild-type strain, were subjected to Southernhybridization analysis (FIG. 6). T. flavus total DNA was prepared forSouthern analysis as follows. Cells from 5 ml overnight TT brothcultures grown at 65° C. were pelleted and resuspended in 900 μl of 25mM pH 8.0 Tris buffer containing 10.3% sucrose, 25 mM EDTA and 1 mg/mllysozyme (Sigma). After a 30 min incubation at 37° C., 250 μl of 500 mMEDTA, 2.5 μl of Proteinase K (20 mg/ml, Sigma), and 140 μl of 10%aqueous lauryl sulfate (SDS) were mixed in. After a second 30 minincubation at 37° C., 150 μl of phenol/chloroform/isoamyl alcohol(25:24:1) was mixed-in until the solution appeared homogeneous. Thesolution was separated into two phases by a 5 min microcentrifugationand 4 μl of RNase (10 mg/ml) was gently mixed into to the upper, aqueousphase and the tube was incubated at 37° C. for another 30 min followedby vigorous mixing of the two phases and a second micro-centrifugation.The aqueous phase (0.9 ml) was transferred to a new tube to which 100 μlof 3M sodium acetate was added and 1 ml of isopropanol. The chromosomalDNA was allowed to precipitate into a globule which was transferred bypipet and washed in a tube containing 70% ethanol and then transferredand dissolved in 100 μl of TE, pH 8.0. Enzymatic digestions for Southernanalyses were performed using between 2 and 8 μl of these DNA solutions.Southern analyses were performed using the materials and methodscontained in the Genius nonradioactive DNA labeling and detection Kit,(Boehringer Mannheim, Indianapolis, Ind.).

All three Leu⁻ transformants were found to have acquired an insertion ofthe size expected for kan^(tr2) in the leuB gene confirming that thekan^(tr2) gene had been inserted into the leuB region of the chromosome,whereas none of the Leu⁺ strains had inserts (lanes 4-6, FIG. 6A). Thedata, therefore, provide evidence for a double recombinationgene-replacement event in which the kan^(tr2) gene was inserted intoleuB in the T. flavus chromosome.

EXAMPLE 7

Stable gene insertion

Stable Leu⁻ Kan^(r) phenotype in T. flavus. Primary transformants of T.flavus with pTG100kan^(tr2), selected for growth on TM plates containing20 μg/ml of Km, were transferred to unsupplemented Tmin agar plates andto Tmin agar plates supplemented with 50 μg/ml L-leucine. The Leu⁻phenotype was scored by careful observation of the two plates and by acomparison of growth to the wild-type strain, after 3-4 days incubationat 65° C.

The transformants carrying gene-replacements were found to be stablykanamycin-resistant and Leu⁻ even after three passages on non-selectiveagar media. The kan^(tr2) gene was not spontaneously amplified ordeleted from the chromosome as shown by unchanged Southern bandingpatterns (not shown). Transformants with pTG100kan^(tr2) appear toefficiently and completely insert kan^(tr2) sometime during the growthof the primary transformants or while being subcultured to produce theDNA for Southern analysis. No evidence of single-crossover intermediateswas obtained in the sample tested. This contrasts with other prokaryoticgene-replacement systems where it is common to isolate single-crossoverintermediates in integration and replacement process. The differencepresumably reflects a more rapid rate of recombination in the T. flavussystem.

EXAMPLE 8

Optimizing temperature

Effect of temperature on pTG100kan^(tr2) transformation efficiencies andbackground growth. During the course of an experiment to observe theeffect of lowering the temperature from 65° C. to 55° C. for selectionof pTG100kan^(tr2) transformants we observed at least a 100-foldincrease in the efficiency of transformation and the elimination of thespontaneous kanamycin resistance growth on the plates. No transformantswere obtained with pTG100kan (containing the non-thermostable kanallele) even after extended incubation periods.

Further reduction of the initial selection temperature to 45° C.produced essentially the same results observed at 55° C. for bothplasmids. The transformants with pTG100kan^(tr2) grew at normal rates:two to three days to appear at 55° C., and up to two weeks to appear at45° C. Again, no transformants appeared with pTG100kan. At 70° C. notransformants or background colonies appeared using either plasmid.

In summary, at 55° C., kanamycin-resistant transformants werereproducibly obtained and easily identified when transformed with aplasmid carrying the thermostabilized marker, kan^(tr2), but not whentransformed with the wild-type marker, kan. Also, transformationefficiencies were at least 100-fold higher at the lower temperaturestested and no background growth appeared.

EXAMPLE 9

Thermostable Mutant Selection

Temperature shifting--selection for more highly temperature-resistantmutants. Although transformants of T. flavus with pTG100kan^(tr2) grewwell in subculture at 55° C., they could not grow at 65° C. In order tosee if mutations that would make the strain able to grow stably athigher temperatures could be selected in the kan^(tr2) transformants,cells from the 55° C. kan^(tr2) transformants were plated at highdensity on agar medium containing kanamycin and incubated at 65° C.Spontaneous mutants that survived the temperature shift appeared at afrequency of one in 10⁻⁶ and could be distinguished from the backgroundgrowth by their larger size. Plasmid pTG100kan^(tr2) transformants grewnormally in subculture on kanamycin-supplemented agar at 65° C. and weredesignated as Kan^(tr2+).

It will be possible to take T. flavus cells which contain kan^(tr2) inthe chromosome and treat with a mutagen to increase the mutationfrequency. Alternatively, a more directed mutagenesis approach could betaken to increase the mutant frequency of kan^(tr2) specifically.Plasmid DNA could be mutated in E. coli before tranformation into T.flavus by treating the cells with hydroxyulamine. In this approach, E.coli cells harboring pTG100kan^(tr2) are subjected to hydroxylamine at aseries of different concentrations. Hydroxylamine causes mutationspreferentially on plasmid molecules in E. coli (Miller, 1972). Plasmidsprepared from the mutagenized E. coli are then used to transform T.flavus and the Thermus cells are plated under conditions of the firsttemperature shift.

Another directed mutagenesis approach utilizes the error-prone PCRtechnique (Spee et al., 1993, Nucleic Acids Res. 21:777-8) In thisapproach, the kan^(tr2) (or other gene of interest) is mutagenized priorto cloning into the gene replacement vector. The kan^(tr2) gene ismutagenized by using the primers which were previously used to clone itinto the NcoI site. Two PCR reactions are run, one with either dA or dGpresent in reduced amounts in the amplification mix. This increases theTaq DNA polymerase error rate and introduces mutations. Nucleotideanalog dl is optionally added to increase the mutation frequencyfurther. This mixture is then cloned as previously described into theNcoI site of pTG100 (or if a second gene is being introduced andmutagenized into pTG100kan^(tr2) sc) and directly used to to transform Tflavus, or can be used to transform E. coli, pooled, and then used toretransfrom Thermus.

Linkage of the Kan^(tr2+) and Leu⁻ phenotypes. In order to determinewhether any of the mutations leading to the Kan^(tr2+) phenotype werelinked to leuB we retransformed the mutant DNA into wild-type T. flavus.This would reveal whether the mutations were likely to be located in ornear to the kan^(tr2) gene (linked), or far from the kan^(tr2) gene(unlinked). Total DNA was isolated from the Kan^(tr2+) Leu⁻ mutants andused to transform wild-type T. flavus at 55° C. Primary transformantswere selected at 55° C. and screened for kanamycin-resistance at 65° C.(i.e., the Kan^(tr2+) phenotype) and for Leu⁻. The results showed thatin one out of ten of the mutants, the Kan^(tr2+) phenotype was 100%linked to the Leu⁻ phenotype in a sample of 50 transformants. The othernine out of ten transformants showed lower degrees of linkage suggestingthat the mutation was further away from the kan^(tr2) gene.

EXAMPLE 10

Multiple Gene Insertion

Insertion of multiple exogenous genes. The following describes anexample of the instant invention which could be used to insert a second(or multiple) exogenous genes into the Thermus chromosome, utilizing theselectability of kan^(tr2) to insert a gene which may not have aselection or which may not operate at the growth temperature of theThermus strain used.

In this example, a recombinant molecule is made which contains the geneof interest cloned into either the upstream NcoI site of pTG100kan^(tr2)cs or the downstream NcoI site of pTG100kan^(tr2) sc or a polylinkerwhich is inserted at one of these sites. One example of doing this is touse plasmid pTG100kan^(tr2) cs is digested with endonuclease NcoI,filled-in with the DNA polymerase Klenow fragment, and dephosporylatedwith Calf-intestinal alkaline phosphatase. The gene of interest issimilarly made blunt-ended, only without phosphorylation, and ligatedinto the filled-in NcoI site. A recombinant molecule with the fragmentin the desired orientation is used to transform T. flavus and selectionfor kanamycin resistance at 55° C. can be used as previously describedfor the kan^(tr2) gene. If the gene of interest is placed upstream ofkan^(tr2), expression of kan^(tr2) indicates that the gene of interesthas likely been transcribed also. Once transformants have been obtained,selection on kanamycin media is no longer required since genereplacements are stable.

EXAMPLE 11

Monitoring Expression

Monitoring expression of gene replacement products. The followingdescribes an example of the instant invention which could be used tomonitor expression levels of genes inserted into the chromosome ofThermus. During a thermostabilization process, several types of mutantscan be generated which allow selection of an increased level of activityat higher temperatures. If a gene, such as kan^(tr2) is used, selectionat higher temperatures could mean that the thermostability of theprotein has increased, or could mean that the expression level of thegene has increased. Mutants need to be individually analyzed todetermine if a thermostability mutation or some other mutation has beenobtained.

In this example, a reporter gene which can be easily assayed, such asone with a β-galactosidase activity, is inserted downstream of the geneof interest can be used to screen away mutations which effect expressionlevels. One such gene is tbg, which has been cloned from T. aquaticusand which produces a thermostable broad specificity β-galactosidaseenzyme, Tbg. Tbg catalyzes the hydrolysis of a wide range of glycolyticcompounds including compounds which can be easily assayed such as5-bromo-4-chloro-3-indolyl-galactopyranoside (X-Gal), and5-bromo-4-chloro-3-indolyl-glucopyranoside (X-Glu) which form blueprecipitates when hydrolyzed and o-nitrophenyl-β-D-galactopyranoside(ONPG) and o-nitrophenyl-β-D-glucopyranoside which can be quantitatedspectrophotometrically.

Plasmid pTAQL2 is is used to PCR amplify a fragment containing tbg. Theprimers are designed to include the tbg ribosomal binding site and startcodon and to have NcoI sites at each end after amplification. The PCRproduct is digested with NcoI and ligated into a filled-in NcoI site inpTG100kan^(tr2) sc. Insertion into the chromosome of Thermus will allowthe tbg gene to be placed under control of the leucine promoter so thatlevels of expression can be easily monitored using one of the assayablesubstrates described above according to Miller (Miller, 1972). Bysupplementing the agar media with X-gal or X-glu (approximately 2 ml ofa 4% solution in n-n-dimethylformamide per liter of media), one canvisualize expression levels on a plate by observing how blue theindividual colonies are. The expression levels can be quantitated usinga substrate such as ONPG and assayed according to Miller.

Derivatives of this plasmid can be made which contain a polylinkerupstream of kan^(tr2) and the tbg inserted downstream to moniterexpression levels. This system is used to optimize expression from theleucine promoter or to screen for mutants in a temperature shiftexperiment which have an increased activity of the desired gene but notof the downstream tbg.

EXAMPLE 12

Chromosomal Transfer

Transferring to other strains of Thermus

The methods and compositions of the instant disclosure allow for thepossible use in the transferring altered chromosomes from one Thermusspecies to another.

In order to have a broader range of temperatures with which tothermostabilize potential exogenous proteins, transferring the genereplacement event to other strains of thermus could be readilyaccomplished by chromasomal transfer. (Koyama et al. 1986) Because ofits high transformation frequency for exogenous DNA sequences, Thermusflavus is the best starting point for chromosomal insertion. Onceaccomplished in flavus the transformation frequency is relatively highwith chromosomal transfers to other related thermus strains withdifferent optimum temperatures for growth.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1707 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 262..810                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGTACCGGGAGGGTCCCTGGAGCCGGGTGGGGATGGTGGTGGGGGCCACCTACCCGGGGG60                CCGTGGCTCGGGTGCGGGAAAGGGCGCCCCACGCCCCCCTCCTCCTCCCCGGCGTGGGGG120               CCCAGGGGGGGAGGCCCTCAAGGGGGAGGGGCTTCTTTTCGCGGCGAGCCGGGCCCTCTA180               CTACCCTGGGGGAAGGCCGGACCTAAAGGCCGCCCTGGAGGCGGCGGAGGCCCTCTTGAA240               GGCTCTGGTAGAGTAGGGGGGATGGACGTCCTGGAGCTTTACCGGAGGACG291                        MetAspValLeuGluLeuTyrArgArgThr                                                1510                                                                          GGGGCTCTTCTAGAGGGCCACTTCCTCCTGCGCTCGGGGATGCACTCC339                           GlyAlaLeuLeuGluGlyHisPheLeuLeuArgSerGlyMetHisSer                              152025                                                                        CCCTTCTTTTTGCAGTCGGCGGCCCTCCTCCAGCATCCCCTTTACGCC387                           ProPhePheLeuGlnSerAlaAlaLeuLeuGlnHisProLeuTyrAla                              303540                                                                        GAGGCCGTGGGGGAGGCTTTGGGAAAGCTCTTTGAGGACGAGAAGGTG435                           GluAlaValGlyGluAlaLeuGlyLysLeuPheGluAspGluLysVal                              455055                                                                        GACTTCGTCATCGCCCCGGCCATCGGGGGCGTGGTCCTTTCCTTCGTG483                           AspPheValIleAlaProAlaIleGlyGlyValValLeuSerPheVal                              606570                                                                        GTGGCGAAGGCCTCGGGCCCGGGCCCTCTTCGCCGAGAAGGACGGAAG531                           ValAlaLysAlaSerGlyProGlyProLeuArgArgGluGlyArgLys                              75808590                                                                      GGGAGGGATGCTCATCCGCAAGGGGCTCACCGTGAACCCGGGCGACGC579                           GlyArgAspAlaHisProGlnGlyAlaHisArgGluProGlyArgArg                              95100105                                                                      TTCTTGGCGGTGGAGGACGTGGTAACCACCGGGGAGAGCGTCCGCAAG627                           PheLeuAlaValGluAspValValThrThrGlyGluSerValArgLys                              110115120                                                                     GCGATCCGGGCGGCGGAGGCCCGGGGCGGGGTTTTGGTGGGCGTGGGG675                           AlaIleArgAlaAlaGluAlaArgGlyGlyValLeuValGlyValGly                              125130135                                                                     GCCATCGTGGACCGGAGCGGGGGCAGGGCGGCCTTCGGCGTGCCCTTC723                           AlaIleValAspArgSerGlyGlyArgAlaAlaPheGlyValProPhe                              140145150                                                                     CGCGCCCTCCTCGCCTTGGAGGTTCCCCAGTATCCCGAGGAGGCCTGC771                           ArgAlaLeuLeuAlaLeuGluValProGlnTyrProGluGluAlaCys                              155160165170                                                                  CCCCTCTGCCGGGAGGGGGTGCCCTTGGAGGAGGTTTAGGGTGCGC817                             ProLeuCysArgGluGlyValProLeuGluGluVal                                          175180                                                                        TTCCTCGCTGCCCTTCTTCTCGGCCTTTTCTCCCTGGCCCTCGCGGCCCCGGAGGAGGCC877               GCGAGGGAGACCGTCGCCCGGTGGCTCAGGGGGGAGCTCTCCCCGAGCCTCGAGGAGGTC937               CTTAGGGCCCCTCCGGAGGAGGCCCCGAGGCTCCTCGAGCGTTCGCCCTCTTCCCCCCGC997               CCCCCGATGGGCTTACCGTCAACCTGGAAAGCCCCGAGGTGGAGGGGAACCGGGTCTCCT1057              TCCCGGCCGCCCTCGGGGAGGAGGTGGGGGAGGTGGTGGTGGTCCTGGAAGGGGGGGAGG1117              CCAGGCGGGTCTACTTCCGCCCCGAGGCTCGGGTGCCCGCCTACCTCCTCACGCCCCTCG1177              CGGGCTTTGGGTTTTTCCTCCTCTCCCTCTTTTGGGTCTTCCTCCTCCTCAGGCCCTCCC1237              CCTTCCGGGCCTGGCTTCTTGAGGCCTGGGCCTTGGTCCGGTCCCAGAGGGGCCTTTACC1297              TCTTCACCAACCTCTTCCTCTACGGCCTATTCGCCCTGGGGAGCCTTCTCGCCTACGCCA1357              TGCCCGAGCTCGCCCGGGCGGTGCAGGTCCTCTTCGGGGGCGCCTTGGAGGCCATCGGCC1417              TCCAGGAGGCGGTGGGGAAGGGCGTTTTGGTCCTCGCTGGGGTCATCTTTCACTGGAATT1477              TCAGCCAGGGGCTTTTCCTCACAGGGCTCCTTCCCGCCTTGCTCTTGGGGGTTCCTGTGC1537              TCCTCCTCAACGCCCTCCGCTACTTCGCTTCGTTCGCCCTCTCCCCGGCCCTTCTGGGAA1597              GCGCCTTCCTCTTCCACCTGCCCACCCTTCTTTTGGAGCTTCAGGCCTACATCCTCGTCA1657              CTTCGGCGGGCTCGTCCTCCTCGCCCGGGTGGCCGGGGGGCAGGGGTACC1707                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAspValLeuGluLeuTyrArgArgThrGlyAlaLeuLeuGluGly                              151015                                                                        HisPheLeuLeuArgSerGlyMetHisSerProPhePheLeuGlnSer                              202530                                                                        AlaAlaLeuLeuGlnHisProLeuTyrAlaGluAlaValGlyGluAla                              354045                                                                        LeuGlyLysLeuPheGluAspGluLysValAspPheValIleAlaPro                              505560                                                                        AlaIleGlyGlyValValLeuSerPheValValAlaLysAlaSerGly                              65707580                                                                      ProGlyProLeuArgArgGluGlyArgLysGlyArgAspAlaHisPro                              859095                                                                        GlnGlyAlaHisArgGluProGlyArgArgPheLeuAlaValGluAsp                              100105110                                                                     ValValThrThrGlyGluSerValArgLysAlaIleArgAlaAlaGlu                              115120125                                                                     AlaArgGlyGlyValLeuValGlyValGlyAlaIleValAspArgSer                              130135140                                                                     GlyGlyArgAlaAlaPheGlyValProPheArgAlaLeuLeuAlaLeu                              145150155160                                                                  GluValProGlnTyrProGluGluAlaCysProLeuCysArgGluGly                              165170175                                                                     ValProLeuGluGluVal                                                            180                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAGCCATGGCCATTTAAAAAGGGAATGAG29                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GAGGCCATGGTTCAAAATGGTATG24                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AATCTAAAATTATCTGAAAAGGGAATGAGAATAGT35                                         (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CACTATTCTCATTCCCTTTTCAGATAATTTTAGATT36                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CATACCATTTTGAACGATGACCTC24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAGGTCATCGTTCAAAATGGTATG24                                                    __________________________________________________________________________

What we claim is:
 1. A method of evolving thermostable proteins whichcomprises subjecting host cells to elevated temperatures for selectionof mutated thermostable proteins in a step-wise fashion, where said hostcells were produced by growing host cells and selecting for a stablehost cell transformant, where the host cells have been transformed witha vector, where said vector transforms the host cell by inserting atargeting DNA sequence into a targeted chromosomal DNA sequence of athermophilic microorganism, where said insertion results in a benignmutant phenotype, which targeting DNA sequence contains an exogenous DNAsequence which encodes an exogenous protein, which exogenous DNAsequence will recombine into the targeted DNA sequence, and theexpression of said exogenous protein from said exogenous DNA sequence isregulated by the chromosome.
 2. The method of claim 1 where theexogenous DNA sequence encodes a kan gene.
 3. The method of claim 1where the targeting DNA sequence and the targeted chromosomal DNAsequence is a leuB gene.
 4. The method of claim 1 where the targetingDNA sequence and the targeted chromosomal DNA sequence is a pyrE gene.5. A host cell which has been produced by the method of claim 1.